The goal of this research effort is to understand how various types of white blood cells recognize and respond to the presence of a microorganism or cancer cell in the body, or inappropriately recognize a normal component of the body (an auto-antigen). Our experiments are designed to provide a detailed understanding of how the substances (antigens) making up microorganisms, cancer cells, or normal self-components, are made visible to the defending cells of the innate (anti-unspecific) or adaptive (antigen-specific) limbs of the host defense system and how recognition of infections, malignant cells, or adjuvants is linked through complex cell-cell interactions to the induction of protective or self-destruction effector responses. During the past year we extended our study combining intravital imaging, in vitro migration analysis, and flow cytometry to explore the role of the lipid-signaling pathway involving S1P and its receptors S1P1 and S1P2 in control of osteoclast genesis. Osteoclasts are large multinucleate myeloid cells responsible for bone resorbtion. They form by fusion of bone surface-adherent osteoclast precursors that are cells in the monocytoid series. We previously showed that S1P and S1P1 played a role in regulating the efficiency with which osteoclast precursor myeloid cells remained attached to the bone surface, with S1P1 signaling promoting the return of the precursors to the blood before maturation occurs. We now have evidence that S1P2 acts in the opposite fashion, promoting chemorepulsion and augmenting the egress of the precursors from the blood and movement towards the bone surface. However, S1P2 operates in a very different range of S1P concentrations from S1P1, allowing these two receptors and the gradient of S1P that exists between bone surface and blood to finely regulate the rate of movement of osteoclast precursors to and from the bone surface. This study thus provides novel insight into a pathway with a major role in the balance of bone formation and destruction and points to a new target for interference with pro-osteoporotic processes. We are using our 2 photon intravital imaging technology on a range of other projects investigating the intersection of the immune system with vaccines, infectious antigen sources, and dying cells in tissues. We are extending our earlier studies on the chemokine control of CD4-CD8 T cell interactions to better understand how these cell types collaborate in lymphoid tissue to generate optimal primary and memory cell-mediated immune responses. Our recent work suggests that different dendritic cells initially present antigen to the majority of CD4 vs. CD8 T cells, raising questions about where and when during the response the two cell types interact with the same antigen presenting cell. We have also acquired data on the role of Tregs in controlling effector vs. central memory cell formation, the site(s) of delivery of TLR-conjugate vaccines to diverse dendritic cell populations in draining lymph nodes, and on the differential location of nave vs. memory cells in lymphoid tissues. The latter studies have also led to exciting new data on the highly localized positioning of a series of innate lymphoid populations within lymph nodes near to the subcapsular sinus. Macrophages lining this sinus play a critical first line defensive role against the spread of lymph-borne pathogens and these lymphoid effectors appear pre-positioned to back up this initial defense against systemic spread of infections by receiving signals generated by inflammasome activation of the macrophages and producing cytokines such as IFN-gamma that augment the anti-microbial activities of the pathogen-sensing macrophages. Interference with this communication leads to systemic spread of the infection. We have also expanded our imaging technologies by developing methods that permit performing 7 or 8 color fluorescent immunohistochemistry on lymphoid and other tissues, collecting tiled images across entire tissue sections, and computationally analyzing the data to assign multiple stains to specific cells. This method has allowed us to fully characterize all the migratory and resident dendritic cell subpopulations in skin draining lymph nodes of mice and to reveal the complex and heterogeneous, but non-random distribution of these various dendritic cell subsets. It has also permitted us to generate hypotheses about which dendritic cell subsets capture antigen delivered in particular ways and to begin to test these models by direct imaging, in concert with an assessment of the T cell subsets with which such antigen-capturing dendritic cells interact. As part of this study, we have developed a new pipeline for image capture, processing, and analysis that permits not only the analysis of cell phenotype and positioning, but also a quantitative assessment of these phenotypically complex cell populations within intact tissue. We call this entire set of analytic tools Histo-cytometry for its similarity in outcome to flow cytometric analysis of dissociated cell populations. This methodology has potential applicability to human and NHP tissues and we have used this method in a collaborative study to examine follicular helper cell (Tfh) in infected monkeys, work that should contribute to the improvement of vaccines. We are also examining the role of various innate receptors in signaling to the cells we are imaging, attempting to integrate the actions of microbial stimuli in our assessment of immune cell behavior in situ. This work includes a broad analysis of NLR-family receptor function. One major outcome of these studies has been the entirely unexpected demonstration that the adapter MAVS, best known for its role in type 1 interferon responses after RIG-I detection of viral RNA, actually plays a critical role in NLRP3 inflammasome activity. Our experiments have revealed that MAVS recruits NLRP3 to mitochondria via a direct protein-protein interaction, and that this activity of MAVS amplifies the activity of NLRP3 with respect to promoting ASC oligomerization and caspase-1 activity. A deficiency in MAVS diminishes the inflammatory response in an acute tubular necrosis model to the same extent as ACS or NLRP3 deficiency, validating the physiological significance of these observations. These findings indicate that the role of adapter proteins is not dictated by their structure alone, but by the nature of the upstream sensing protein with which they interact, as inflammasome activation using agents such as nigericin or poly I:C gives rise to IL-1b and not type 1 interferon in contrast to RIG-I-dependent signaling. We have developed a novel method that permits the rapid screening of control vs. mutant (knock-out) cells for differences in behavior within a tissue site, especially the localized swarming response of neutrophils to sterile tissue damage. Studies using this method have revealed a very specific role for lipid mediators in neutrophil responses to cell death as well as an unsuspected effect of neutrophil aggregation on local tissue matrix structure. These studies are being extended further using 3D culture systems and microfabricated devices that permit precise control of chemoattractant gradient steepness and magnitude, as well as changes in effective concentration over time. Using these 3D systems, we have developed an entirely new picture of the factors controlling persistent migration of myeloid cells. Our new results suggest that temporal rather than spatial sensing plays a crucial role in persistent directional migration of various myeloid cell types, results that fit with two sets of in vivo data that indicate robust amplification loops operating during innate inflammatory processes that would provide the needed rising chemoattractant gradients required for such directed migration in vivo.